relA (PA0934) encodes the ribosome-associated (p)ppGpp synthetase I of Pseudomonas aeruginosa, a long RelA/SpoT homologue (RSH) with an N-terminal hydrolase-like domain, a synthetase domain, and C-terminal TGS, helical and ACT regulatory domains. Activated by uncharged tRNA at stalled ribosomes during amino-acid starvation, RelA transfers pyrophosphate from ATP to GTP/GDP to make pppGpp and ppGpp, the alarmones of the stringent response. In PAO1 the relA mutant makes no (p)ppGpp under serine starvation, whereas the paralog SpoT (PA5338) supplies ppGpp in stationary phase and under other starvations and is the bifunctional enzyme that also degrades it; RelA of beta- and gamma-proteobacteria has lost hydrolase activity. Through (p)ppGpp, RelA influences quorum-sensing signal production, RpoS levels, stress tolerance and virulence.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0008728 GTP diphosphokinase activity | IBA GO_REF:0000033 | ACCEPT | Summary: PAINT IBA from PTN001083175, seeded by E. coli RelA (P0AG20). The synthetase activity is the core function of RelA. Reason: The P. aeruginosa relA mutant makes no (p)ppGpp under amino-acid starvation and multicopy relA restores synthesis, consistent with the conserved RelA synthetase domain. The node (RelA, SpoT and Rel) shares the synthetase. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN001083175 · PTN001083175 SUPPORTS TRANSFER All 23 leaves are long RSHs with a synthetase domain. UniProtKB:P0AG20 · E. coli RelA SUPPORTS TRANSFER Supporting Evidence: PMID:15385461 ppGpp and pppGpp were undetectable in the relA mutant, whereas wild-type PAO1 produced high levels of ppGpp and pppGpp PMID:15385461 We were also able to restore ppGpp and pppGpp production through overexpressing the relA gene on the multicopy vector pUCP18 |
| GO:0008893 guanosine-3',5'-bis(diphosphate) 3'-diphosphatase activity | IBA GO_REF:0000033 | REMOVE | Summary: PAINT IBA of (p)ppGpp hydrolase activity, seeded by E. coli SpoT (P0AG24) at the node that contains both the RelA and the SpoT paralogs. Reason: In beta- and gamma-proteobacteria the ancestral bifunctional Rel duplicated into SpoT, which kept the hydrolase, and RelA, whose HD domain diverged and lost hydrolase activity. Placing the hydrolase IBD on the duplication ancestor propagates it to every RelA leaf, including this one. No hydrolase activity has been reported for P. aeruginosa RelA, and in PAO1 degradation is attributed to SpoT. Propagation Review Root cause: PROPAGATION BAD Failure modes: PSEUDO OR SUBACTIVITY LOSS WRONG ORTHOLOG OR PARALOG Sources checked: PANTHER:PTN001083175 · PTN001083175 SUPPORTS SOURCE BUT NOT TARGET The node spans the RelA/SpoT duplication; the hydrolase is retained only in the SpoT and Rel lineages. UniProtKB:P0AG24 · E. coli SpoT SUPPORTS SOURCE BUT NOT TARGET Supporting Evidence: PMID:21858139 while RelA is clearly more divergent, consistent with its loss of hydrolase activity PMID:21788391 which is synthesized by the enzymes RelA and SpoT and which is also degraded by SpoT |
| GO:0015949 nucleobase-containing small molecule interconversion | IEA GO_REF:0000117 | MODIFY | Summary: ARBA electronic annotation. Formally true (GTP and GDP are converted to (p)ppGpp) but vague. Reason: RelA's process is (p)ppGpp synthesis; GO:0015970 guanosine tetraphosphate biosynthetic process is the specific supported term. Proposed replacements: guanosine tetraphosphate biosynthetic process Supporting Evidence: PMID:15385461 These results indicate that the relA gene is necessary for ppGpp and pppGpp production in P. aeruginosa . |
| GO:0015968 stringent response | IMP PMID:15385461 Pseudomonas aeruginosa relA contributes to virulence in Dros... | ACCEPT | Summary: PseudoCAP IMP; sole seed of the GO:0042594 response to starvation IBD at PTN001083175. Reason: The relA deletion abolishes (p)ppGpp production after serine hydroxamate-induced serine starvation and multicopy relA restores it. RelA synthesises the alarmone that is the stringent response signal, so the gene does the work of the process; this is not an indirect phenotype. Other phenotypes in the paper (virulence, signals, pyocyanin, RpoS) were not annotated. Supporting Evidence: PMID:15385461 The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. |
| GO:0015969 guanosine tetraphosphate metabolic process | IBA GO_REF:0000033 | MODIFY | Summary: PAINT IBA from PTN001083175 (E. coli RelA seed). Correct but general. Reason: RelA only synthesises (p)ppGpp (its hydrolase is inactive), so the biosynthetic child GO:0015970 is the precise term. The metabolic-process term is appropriate at the node, which includes the bifunctional SpoT and Rel. Propagation Review Root cause: NO FAILURE CORE Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN001083175 · PTN001083175 SUPPORTS TRANSFER Metabolic process is correct for the whole node; RelA leaves only synthesise. UniProtKB:P0AG20 · E. coli RelA SUPPORTS TRANSFER Proposed replacements: guanosine tetraphosphate biosynthetic process Supporting Evidence: PMID:15385461 These results indicate that the relA gene is necessary for ppGpp and pppGpp production in P. aeruginosa . |
| GO:0015969 guanosine tetraphosphate metabolic process | IEA GO_REF:0000002 | MODIFY | Summary: InterPro2GO mapping from the RelA/SpoT family signatures. Correct but general. Reason: As for the IBA row with the same term; RelA synthesises but does not degrade (p)ppGpp. Proposed replacements: guanosine tetraphosphate biosynthetic process Supporting Evidence: PMID:15385461 These results indicate that the relA gene is necessary for ppGpp and pppGpp production in P. aeruginosa . |
| GO:0042594 response to starvation | IBA GO_REF:0000033 | MODIFY | Summary: PAINT IBA from PTN001083175, whose sole seed is this gene's PseudoCAP IMP to stringent response. The target appearing in its own WITH/FROM is expected. Reason: True and a sound generalisation for the node (RelA, SpoT and Rel all make (p)ppGpp in response to some starvation), but for RelA the specific supported process is GO:0015968 stringent response, a descendant (via cellular response to starvation) that the gene already carries by IMP. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN001083175 · PTN001083175 SUPPORTS TRANSFER 23 leaves (13 taxa) in the PAINT leaf GAF, RelA and SpoT pairs of beta/gamma-proteobacteria plus Rel of Helicobacter, Geobacter and Bradyrhizobium; all are starvation-responsive (p)ppGpp synthetases. UniProtKB:Q9I524 · relA SUPPORTS TRANSFER The target's own IMP is the seed; expected, not circular. Proposed replacements: stringent response Supporting Evidence: PMID:15385461 The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. PMID:21788391 no (p)ppGpp synthesis could be detected in the relA spoT double mutant under any growth condition tested, including starvation for nutrients such as amino acids, carbon, and iron |
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Download this section (compressed HTML)Q: Does P. aeruginosa RelA retain any residual (p)ppGpp hydrolase activity, or is its HD-like domain purely regulatory as in E. coli RelA?
Experiment: Assay purified P. aeruginosa RelA for (p)ppGpp hydrolysis in vitro, with SpoT as a positive control, and test whether relA complements a spoT-null hydrolase defect.
Type: in vitro enzyme assay
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